title: A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.
abstract: We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.
authors.affiliation: Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. helen.toogood@manchester.ac.uk
authors.forename: Helen S
authors.initials: HS
authors.lastname: Toogood
authors.forename: Anna
authors.initials: A
authors.lastname: Fryszkowska
authors.forename: Martyn
authors.initials: M
authors.lastname: Hulley
authors.forename: Michiyo
authors.initials: M
authors.lastname: Sakuma
authors.forename: David
authors.initials: D
authors.lastname: Mansell
authors.forename: Gill M
authors.initials: GM
authors.lastname: Stephens
authors.forename: John M
authors.initials: JM
authors.lastname: Gardiner
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000475:Alkenes / Q000378:metabolism; D018360:Crystallography, X-Ray; D016972:Enterobacter cloacae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010091:Oximes / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D011485:Protein Binding; D020592:Shewanella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D000475:Alkenes; D010091:Oximes; D010636:Phenols; D010088:Oxidoreductases; C098939:pentaerythritol tetranitrate reductase